Granzyme b protease variants

ABSTRACT

A polypeptide having a serine protease variant of the human granzyme B set forth by SEQ ID NO: 1, wherein the serine protease variant has at least 95% identity to SEQ ID NO: 1 and has a substitution at the position that corresponds structurally or by amino acid sequence homology to position Arg201 of SEQ ID NO: 1, and wherein the serine protease variant has activity to cleave the motif Ile-Glu-Thr-Asp.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 14/346,600,which is a national phase application under 35 U.S.C. §371 ofinternational patent application no. PCT/EP2012/068607, filed Sep. 21,2012, which claims priority to U.S. provisional patent application No.61/538,368, filed Sep. 23, 2011 and European patent application no.11007764.1, filed Sep. 23, 2011. Each of the above documents areincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing as file“US14346600_2014-08-19_SEQ_ID” created on 19 Aug. 2016, and having asize of 24 Kilobytes. The sequence listing contained in this ASCIIformatted document forms part of the specification and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The technology provided herein relates to novel variants of enzymesexhibiting serine protease activity, more specifically to improvedvariants of the serine protease granzyme B; to nucleic acid moleculesencoding said proteases, vectors, host cells containing the nucleicacids and methods for preparation and producing such enzymes;compositions and complexes comprising at least one of the proteases; andmethods for using such enzymes as a part of an immunoprotease, inparticular for the treatment of cancer.

BACKGROUND

In the treatment of tumors, autoimmune diseases, allergies and tissuerejection reactions, it is a disadvantage that the currently availablemedicaments, such as chemotherapeutic agents, corticosteroids andimmunosuppressive agents, have a potential of side effects which issometimes considerable, due to their relative non-specificity. It hasbeen attempted to moderate this by various therapeutical concepts.Especially the use of immunotherapeutic agents is an approach, whichresulted in an increase of the specificity of medicaments, especially intumor treatment.

If the immunotherapeutic agent is an immunotoxin, then a monoclonalantibody (moAb) or an antibody fragment, which has a kinetic affinityfor surface markers of tumor cells, is coupled with a cytotoxic reagent.If the immunotherapeutic agent is an anti-immunoconjugate for thetreatment of autoimmune diseases, tissue rejection reactions orallergies, a structure relevant to pathogenesis or a fragment thereof iscoupled to a toxin component. It has been found that immunotoxins can becharacterized by a high immunogenicity in clinical use. This causes theformation of neutralizing antibodies in the patient, which inactivatethe immunotoxin. Generally, a repeated and/or continuous administrationof the therapeutic agents is unavoidable for long-term curative effects.This is particularly clear in the suppression of tissue rejectionreactions after transplantations, or in the treatment of autoimmunediseases, due to the partly demonstrated genetically causedpredisposition to a pathogenic autoimmune reaction.

The peptidic cell poisons which have been mostly used to date and arethus best characterized are the bacterial toxins diphtheria toxin (DT)(Beaumelle, B. et al. 1992; Chaudhary, V. et al. 1990; Kuzel, T. M. etal. 1993; LeMaistre, C. et al. 1998), Pseudomonas exotoxin A (PE) (FitzGerald, D. J. et al. 1988; Pai, L. H. and Pastan, I. 1998), and theplant-derived ricin-A (Engert, A. et al. 1997; Matthey, B. et al. 2000;O'Hare, M. et al. 1990; Schnell, R. et al. 2000; Thorpe, P. E. et al.1988; Youle, R. J. and Neville, D. M. J. 1980). The mechanism ofcytotoxic activity is the same in all of these toxins despite of theirdifferent evolutionary backgrounds. The catalytic domain inhibitsprotein biosynthesis by a modification of the elongation factor EF-2,which is important to translation, or of the ribosomes directly, so thatEF-2 can no longer bind (Endo, Y. et al. 1987; Iglewski, B. H. andKabat, D. 1975).

In most of the constructs employed to date, the systemic application ofimmunotoxins results in more or less strong side effects. In addition tothe “vascular leak” syndrome (Baluna, R. and Vitetta, E. S. 1997;Schnell, R. et al. 1998; Vitetta, E. S. 2000), thrombocytopenia,hemolysis, renal insufficiency and sickness occur, depending on theconstruct employed and the applied dosage. Dose-dependent and reversibleliver damage could also be observed (Battelli, M. G. et al. 1996;Grossbard, M. L. et al. 1993; Harkonen, S. et al. 1987). In addition tothe documented side effects, the immunogenicity of the constructsemployed to be observed in the use of the immunoconjugates orimmunotoxins is the key problem of immunotherapy (Khazaeli, M. B. et al.1994). This applies, in particular, to the humoral defense against thecatalytic domains employed, such as ricin (HARA) (Grossbard, M. L. etal. 1998), PE (Kreitman, R. J. et al. 2000), or DT (LeMaistre, E. F. etal. 1992). Theoretically, all non-human structures can provoke an immuneresponse. Thus, the repeated administration of immunotoxins andimmunoconjugates is subject to limitations. A logical consequence ofthese problems is the development of human immunotoxins, now named humancytolytic fusion proteins (Rybak, S. et al. 1992).

WO 01/80880 A1 discloses the use of granzyme B as a humanimmunoprotease. The cytotoxic lymphocyte serine proteinase granzyme Binduces apoptosis of abnormal cells by cleaving intracellular proteinsat sites similar to those cleaved by caspases. However, granzyme B has anumber of efficient natural inhibitors that prevent granzyme B-mediatedapoptosis in certain cell types.

Therefore the availability of a human serine protease with improvedapoptotic activity and reduced sensitivity towards activity-inhibitingsubstances would be highly advantageous.

SUMMARY OF THE INVENTION

In a first aspect, embodiments of the disclosure provide polypeptidescomprising a serine protease variant of wildtype human granzyme B,having a modification at one or more positions and showing increasedapoptotic activity compared to wildtype granzyme B and reducedsensitivity to activity-inhibiting substances.

In a further aspect, embodiments of this disclosure relate topolypeptides comprising a serine protease variant of wildtype humangranzyme B as shown in SEQ ID NO: 1, having a modification at one ormore positions selected from the group of positions that correspondstructurally or by amino acid sequence homology to the positions 28and/or 201, or variants, modified forms, homologs, fusion proteins,functional equivalents or functional fragments thereof, wherein saidpolypeptide having a greater apoptotic activity compared to wildtypegranzyme B and reduced sensitivity to activity-inhibiting substances.

In a further aspect, embodiments of this disclosure relate topolypeptides comprising a serine protease having at least 90% identityto amino acids 1-227 of SEQ ID NO: 1, and which, as compared to aminoacids 1-227 of SEQ ID NO: 1, comprises at least one modification at oneor more positions corresponding to position 28 and/or 201, or a modifiedform thereof, wherein said polypeptides having a greater apoptoticactivity compared to wildtype granzyme B and reduced sensitivity toactivity-inhibiting substances.

In still another aspect, embodiments of this disclosure provide nucleicacids encoding polypeptides variants with serine protease activity asdisclosed herein, as well as vectors and host cells comprising suchnucleic acids.

In other aspects, this disclosure relates to compositions comprisingpolypeptide variants as described herein, wherein the compositions maybe useful for, or used in, therapeutical, cosmetic and/or diagnosticapplications. In one advantageous embodiment, the composition is used asa therapeutical composition for the treatment of cancer.

In a further aspect, embodiments of this disclosure relate to methodsfor producing the polypeptide variants in a host cell by transformingthe host cell with a DNA construct, advantageously including a promoterhaving transcriptional activity in the host cell, cultivating thetransformed host cell in a suitable culture medium to allow expressionof said protease and producing the protease. The method may also includerecovering the produced protease.

In an advantageous embodiment of this disclosure, the polypeptide havingserine protease activity has the nucleic acid sequence of SEQ ID NO: 3,SEQ ID NO: 4 or SEQ ID NO: 5 or variants, modified forms, homologs,fusion proteins, functional equivalents or functional fragments thereof.

The polypeptides according to present disclosure can be used forpreparing a medicament for preventing or treating a disease likeallergy, autoimmune reaction, tissue rejection reaction, or chronicinflammation reaction, in particular cancer.

In a further aspect, the disclosure relates to purified complexescomprising a binding structure and a polypeptide according to presentdisclosure, medicaments comprising such a complex in combination with apharmacologically acceptable carrier or diluent and the use of such acomplex for treating a malignant disease, an allergy, autoimmunereaction, tissue rejection reaction, or chronic inflammation reaction,in particular cancer.

Further, embodiments of this disclosure relate generally to the use ofthe polypeptides, compositions and complexes according to the presentdisclosure for the induction of cell death, in particular induction ofcell death by apoptosis. Advantageously, polypeptides, compositions andcomplexes of this disclosure may be used for treating cancer.

Before the disclosure is described in detail, it is to be understoodthat this disclosure is not limited to the particular component parts ofthe devices described or process steps of the methods described as suchdevices and methods may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting. It must be notedthat, as used in the specification and the appended claims, the singularforms “a,” “an” and “the” include singular and/or plural referentsunless the context clearly dictates otherwise. It is moreover to beunderstood that, in case parameter ranges are given which are delimitedby numeric values, the ranges are deemed to include these limitationvalues.

To provide a comprehensive disclosure without unduly lengthening thespecification, the applicant hereby incorporates by reference each ofthe patents and patent applications referenced above, in particular thedisclosure of WO 01/80880 A2 and US 2009/0081185 A1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show examples for fusion proteins including granzyme Bvariants.

FIGS. 2A-B show the expression of granzyme B mutants in a Coomassiestained SDS-PAGE gel and Western Blot.

FIG. 3 shows the determination of endogenous Serpin B9 in different celllines via flow cytometric analysis. Mean Fluorescence Intensity (MFI) ofdetected Serpin B9 is compared between Hodgkin lymphoma derived cellslines (L428, K562, L1236) compared to control cell lines (L540, Jurkat).

FIGS. 4A-B show the determination of Serpin B9 in different cell linesvia western blot analysis. In FIG. 4A, a western blot of Serpin B9 fromHodgkin lymphoma derived cell line (L428, L1236) is compared to control(L540, Jurkat). In FIG. 4B, a western blot of Serpin B9 from humanpromyelocytic leukemia (HL60) cell line is compared to positive control(recombinant PI9 from E. coli).

FIG. 5 is a diagram showing the specific binding of Gb-Ki4(scFv) mutantsto target cells.

FIG. 6 illustrates the proteolytic activity of granzyme B variants in adiagram.

FIGS. 7A-B show the results of an apoptosis assay of Gb-Ki4(scFv)variants on target cell line L1236: flow cytomery (Dotplots).

FIGS. 8A-C show diagrams with the results of an apoptosis assay ofGb-Ki4(scFv) variants on P19⁺ L1236 target cells.

FIGS. 9A-C show the results of an apoptosis assay of Gb-Ki4(scFv)variants on L428 target cell line.

FIGS. 10A-B show an amino acid sequence (FIG. 10A) and a nucleic acidsequence (FIG. 10B) of human wildtype granzyme B (SEQ ID NO: 1, SEQ IDNO: 2).

FIGS. 11A-C show nucleic acid sequences of improved granzyme B variants(SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5).

FIG. 12A-D show amino acid sequences of improved granzyme B variants(SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9).

FIG. 13 shows a protein sequence alignment of consensus sequence forwildtype granzyme B comprising different alleles as well as thepositions for modifications.

FIG. 14 shows a nucleic acid sequence alignment of consensus sequencefor of human granzyme B wildtype.

FIG. 15 is a bar graph showing the results of an apoptosis assay ofGb-Ki4(scFv) mutants on P19⁺ K562 target cell line.

FIG. 16 is a bar graph showing the result of a Caspase 3/7 assay afterincubation of CD30 positive target cell lines with Gb-Ki4(scFv) andGb-Ki4(scFv) R201K.

FIGS. 17A-E show result of ex vivo experiments on primary material ofleukemic patients using Gb-H22(scFv) and mutants. FIG. 17A is a graphshowing results from an Annexin V assay after 24 hours, which depictspercent apoptosis of primary leukemia cells when using the differentserine protease variants. FIG. 17B is a graph showing results from anXTT assay of primary leukemia cells, which measures percent viabilityafter 72 hours of exposure to a cytolytic fusion protein when usingdifferent serine protease variants. FIG. 17C is a photograph depicting awestern blot of primary leukemia cells from FIG. 17A and FIG. 17B formeasurement of Serpin B9 over time. FIG. 17D is a graph showing resultsfrom another Annexin V assay using primary cells from another leukemiapatient, which depicts percent apoptosis when using different serineprotease variants. FIG. 17E is a photograph depicting a western blot ofthe primary cells used in FIG. 17D for measurement of Serpin B9 overtime.

FIGS. 18A-B show changes in tumor size in mice after cytolytic fusionprotein treatment when using serine protease variant R201K, whereGb-Ki4(scFv) R201K treatment is depicted as black triangles,Gb-Ki4(scFv) is depicted as grey triangles, and Gb-H22(scFv) R201K isdepicted as grey squares.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are variants of the serine protease granzyme B and theuse thereof for the treatment of various diseases.

In particular, serine protease variants according to the presentdisclosure showing increased proteolytic activity inducing cellapoptosis (apoptotic activity) compared to wildtype granzyme B enzymesand a reduced sensitivity to activity-inhibiting substances like SerpinB9 These characteristics make them specifically useful as a part of apowerful cytolytic fusion protein, in particular as a part of a complexaccording to the present disclosure. In particular, the serine proteasevariants according to the present disclosure showing a greater apoptoticactivity compared to wild type granzyme B due to the reduced sensitivityto the activity-inhibiting substance Serpin B9.

The present disclosure reveals enzymes with an amino-acid sequencederived from the amino acid sequence shown in SEQ ID NO:1 or variants,modified forms, homologs, fusion proteins, functional equivalents orfunctional fragments thereof, having one or more modifications at one ormore positions selected from the group of positions that correspondstructurally or by amino acid sequence homology to the positions 28and/or 201.

The term “modified form” or “variant” means that the enzyme has beenmodified from its original form (parent/wildtype, wt) but retains thesame enzymatic functional characteristics as that of human wildtypegranzyme B.

The term “fusion proteins” comprises all proteins derived from the humanwildtype granzyme B or any variant thereof by covalently fusingadditional amino-acid sequences at the C- and/or N-terminus. The sourceand composition of the additional amino-acid sequence is either naturalfrom any living organisms or virus or unnatural. In particular, thefusion protein may be a “recombinant” polypeptide, which is definedeither by its method of production or its structure. In reference to itsmethod of production, e.g., a product made by a process, the processinvolved uses of recombinant nucleic acid techniques. In reference tostructure, recombinant polynucleotides or polypeptides contain sequencesfrom different sources. In particular, it encompasses polypeptides madeby generating a sequence comprising two or more fragments which are notnaturally contiguous or operably linked to each other. Thus, forexample, products made by transforming cells with any unnaturallyoccurring vector are encompassed.

The term “functional fragment” or “effective fragment” means a fragmentor portion of the human wildtype granzyme B or derivative thereof thatretains about the same enzymatic function or effect.

The term “homologous polypeptide” according to the present disclosurecomprises any enzyme with a sequence identity of at least 70% orpreferably at least 80%, 85%, 90%, 95%, 97% or 99% to the human wildtypegranzyme B.

The term “polynucleotide” corresponds to any genetic material of anylength and any sequence, comprising single-stranded and double-strandedDNA and RNA molecules, including regulatory elements, structural genes,groups of genes, plasmids, whole genomes and fragments thereof.

The term “position” in a polynucleotide or polypeptide refers tospecific single bases or amino acids in the sequence of thepolynucleotide or polypeptide, respectively.

The term “granzyme B variants” means any granzyme B molecule obtained bysite-directed or random mutagenesis, insertion, deletion, recombinationand/or any other protein engineering method, which leads to granzyme Bthat differ in their amino acid sequence from the human wildtypegranzyme B. The terms “wildtype granzyme B”, “wildtype enzyme”, or“wildtype” in accordance with the disclosure describe a serine proteaseenzyme with an amino acid sequence found in nature or a fragmentthereof.

The term “isolated” describes any molecule separated from its naturalsource.

The term “mutation” refers to the substitution or replacement of singleor multiple nucleotide triplets, insertions or deletions of one or morecodons, homologous or heterologous recombination between differentgenes, fusion of additional coding sequences at either end of theencoding sequence, or insertion of additional encoding sequences or anycombination of these methods, which result in a polynucleic acidsequence encoding the desired protein. Thus, the term “mutations” alsorefers to all of the changes in the polypeptide sequence encoded by thepolynucleic acid sequence modified by one or more of the above describedchanges. Amino acid residues are abbreviated according to the followingTable 1 either in one- or in three-letter code.

The term “nucleic acid molecule” or “nucleic acid” is intended toindicate any single- or double stranded nucleic acid molecule of cDNA,genomic DNA, synthetic DNA or RNA, Peptide nucleic acid (PNA) or LNAorigin

The term “stringent conditions” relates to conditions under which aprobe will hybridize to its target subsequence, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetsequences are generally present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Typically, stringent conditions will be thosein which the salt concentration is less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g. 10to 50 nucleotides) and at least about 60° C. for longer probes.Stringent conditions may also be achieved with the addition ofdestabilizing agents, such as formamide and the like.

The term “variant of the nucleic acid molecule” refers herein to anucleic acid molecule which is substantially similar in structure andbiological activity to a nucleic acid molecule according to one of theclaimed sequences.

The term “homologue of the nucleic acid molecule” refers to a nucleicacid molecule the sequence of which has one or more nucleotides added,deleted, substituted or otherwise chemically modified in comparison to anucleic acid molecule according to one of the claimed sequences,provided always that the homologue retains substantially the samebinding properties as the latter.

The term “derivative” as used herein, refers to a nucleic acid moleculethat has similar binding characteristics to a target nucleic acidsequence as a nucleic acid molecule according to one of the claimedsequences

The term “immunotoxin” refers to a complex comprising a targetingportion linked to a bacterial/plant toxin.

The term “cytolytic fusion protein” or “human cytolytic fusion protein”refers to a complex of a targeting portion linked to a human enzyme.

The term “immunoprotease” refers to a complex of a targeting portionlinked to a human protease.

The term “modification” as used herein, refers for example tosubstitutions, insertions or deletions of amino acid residues atspecific positions in an amino acid sequence as well as thephosphorylation, acetylation like palmitoylation, methylation,sulphation, glycosylation, lipidation like isoprenylation,farnesylation, attachment of a fatty acid moiety, glypiation and/orubiquitinylation of specific positions on the polypeptide, orcombinations thereof.

TABLE 1 Amino acid abbreviations Abbreviations Amino acid A Ala AlanineC Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F PhePhenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K LysLysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline QGln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine WTrp Tryptophan Y Tyr Tyrosine

Mutations or variations are described by use of the followingnomenclature: position; substituted amino acid residue(s). According tothis nomenclature, the substitution of, for instance, an alanine residuefor a glycine residue at position 20 is indicated as 20G. When an aminoacid residue at a given position is substituted with two or morealternative amino acid residues these residues are separated by a commaor a slash. For example, substitution of alanine at position 30 witheither glycine or glutamic acid is indicated as 20G/E, or 20G, 20E.

Furthermore, the following nomenclature could also be used: amino acidresidue in the protein scaffold; position; substituted amino acidresidue(s). According to this nomenclature, the substitution of, forinstance, an alanine residue for a glycine residue at position 20 isindicated as Ala20Gly or A20G, or 20G. The deletion of alanine in thesame position is shown as Ala20* or A20*. The insertion of an additionalamino acid residue (e.g. a glycine) is indicated as Ala20AlaGly orA20AG. The deletion of a consecutive stretch of amino acid residues(e.g. between alanine at position 20 and glycine at position 21) isindicated as A(Ala20-Gly21) or A(A20-G21). When a sequence contains adeletion in comparison to the parent protein used for numbering, aninsertion in such a position (e.g. an alanine in the deleted position20) is indicated as *20Ala or *20A. Multiple mutations are separated bya plus sign or a slash.

For example, two mutations in positions 20 and 21 substituting alanineand glutamic acid for glycine and serine, respectively, are indicated asA20G+E21S or A20G/E21S. When an amino acid residue at a given positionis substituted with two or more alternative amino acid residues theseresidues are separated by a comma or a slash. For example, substitutionof alanine at position 30 with either glycine or glutamic acid isindicated as A20G,E or A20G/E, or A20G, A20E. When a position suitablefor modification is identified herein without any specific modificationbeing suggested, it is to be understood that any amino acid residue maybe substituted for the amino acid residue present in the position. Thus,for instance, when a modification of an alanine in position 20 ismentioned but not specified, it is to be understood that the alanine maybe deleted or substituted for any other amino acid residue (i.e. any oneof R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).

The terms “conservative mutation”, or “conservative substitution”,respectively, refer to an amino acid mutation that a person skilled inthe art would consider a conservative to a first mutation.“Conservative” in this context means a similar amino acid in terms ofthe amino acid characteristics. If, for example, a mutation leads at aspecific position to a substitution of a non-aliphatic amino acidresidue (e.g. Ser) with an aliphatic amino acid residue (e.g. Leu) thena substitution at the same position with a different aliphatic aminoacid (e.g. Ile or Val) is referred to as a conservative mutation.Further amino acid characteristics include size of the residue,hydrophobicity, polarity, charge, pK-value, and other amino acidcharacteristics known in the art. Accordingly, a conservative mutationmay include substitution such as basic for basic, acidic for acidic,polar for polar etc. The sets of amino acids thus derived are likely tobe conserved for structural reasons. These sets can be described in theform of a Venn diagram (Livingstone C. D. and Barton G. J. (1993)“Protein sequence alignments: a strategy for the hierarchical analysisof residue conservation” Comput. Appl Biosci. 9: 745-756; Taylor W. R.(1986) “The classification of amino acid conservation” J. Theor. Biol.119; 205-218). Conservative substitutions may be made, for example,according to the table below which describes a generally accepted Venndiagram grouping of amino acids.

TABLE 2 Venn diagram grouping amino acids Set Sub-set HydrophobicF W Y H K M I L V Aromatic F W Y H A G C Aliphatic I L V PolarW Y H K R E D C S Charged H K R E D T N Q Positively H K R chargedNegatively E D charged Small V C A G S P T N D Tiny A G S

The term “plasmid”, “vector system” or “expression vector” means aconstruct capable of in vivo or in vitro expression. In the context ofthe present disclosure, these constructs may be used to introduce genesencoding enzymes into host cells.

The term “host cell” in relation to the present disclosure includes anycell that comprises either the nucleic acid molecule or an expressionvector as described above and which is used in the recombinantproduction of an enzyme having the specific properties as defined hereinor in the methods of the present disclosure.

“Percent sequence identity”, with respect to two amino acid orpolynucleotide sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 80% amino acid sequence identity means that 80% of the amino acidsin two optimally aligned polypeptide sequences are identical. Percentidentity can be determined, for example, by a direct comparison of thesequence information between two molecules by aligning the sequences,counting the exact number of matches between the two aligned sequences,dividing by the length of the shorter sequence, and multiplying theresult by 100. Readily available computer programs can be used to aid inthe analysis, such as ALIGN, Dayhoff, M. O. in “Atlas of ProteinSequence and Structure”, M. O. Dayhoff et., Suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., which adapts the localhomology algorithm of Smith and Waterman (1981) Advances in Appl. Math.2:482-489 for peptide analysis. Programs for determining nucleotidesequence identity are available in the Wisconsin Sequence AnalysisPackage, Version 8 (available from Genetics Computer Group, Madison,Wis.) for example, the BESTFIT, FASTA and GAP programs, which also relyon the Smith and Waterman algorithm. These programs are readily utilizedwith the default parameters 5 recommended by the manufacturer anddescribed in the Wisconsin Sequence Analysis Package referred to above.An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol. 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. Likewise, computer programs for determiningpercent homology are also readily available.

It is also understood that the present disclosure comprises allmolecules that are derived from the polynucleotides of the disclosureand all variants thereof described in this application, byposttranslational processing compared to the genetically encoded aminoacid sequence. These posttranslational modifications comprise, but arenot limited to, proteolytic cleavage of N-terminal sequences such asleader and/or pro-sequences, proteolytic removal of C-terminalextensions, N- and/or O-glycosylation, lipidation, acylation,deamidation, pyroglutamate formation, phosphorylation and/or others, orany combination thereof, as they occur during production/expression bythe native host or any suitable expression host. Thesepost-translational modifications may or may not have an influence on thephysical or enzymatic properties of the enzymes as explored herein.

In preferred embodiments of the present disclosure, the modified humangranzyme B has a substitution at one or more of the positions 28 and/or201, relative to the numbering of human wildtype granzyme B given in SEQID NO: 1. These positions are characterized in that mutagenesis of theenzyme at these positions leads to improvement in the desired enzymecharacteristics.

In yet a further aspect, the disclosure relates to a nucleic acidmolecule and to the use of a nucleic acid molecule selected from thegroup consisting of

-   -   a) a nucleic acid molecule encoding a polypeptide according to        the present disclosure;    -   b) a nucleic acid molecule encoding for a modified form of the        polypeptide according to the present disclosure, preferably in        which one or more amino acid residues are conservatively        substituted;    -   c) a nucleic acid molecule that is a fraction, variant,        homologue, derivative, or fragment of the nucleic acid molecule        presented as SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5;    -   d) a nucleic acid molecule that is capable of hybridizing to any        of the nucleic acid molecules of a)-c) under stringent        conditions    -   e) a nucleic acid molecule that is capable of hybridizing to the        complement of any of the nucleic acid molecules of a)-d) under        stringent conditions    -   f) a nucleic acid molecule having a sequence identity of at        least 95% with any of the nucleic acid molecules of a)-e) and        encoding for a serine protease,    -   g) a nucleic acid molecule having a sequence identity of at        least 70% with any of the nucleic acid molecules of a)-f) and        encoding for a serine protease,    -   h) or a complement of any of the nucleic acid molecules of        a)-g).

A nucleotide or nucleic acid is considered to hybridize to one of theabove nucleotides if it is capable of hybridizing under conditions ofmedium stringency, more preferably high stringency, even more preferablyunder very high stringency conditions.

The nucleic acid molecule of the present disclosure may comprisenucleotide sequences that encode for SEQ ID NO:3, or an effectivefragment thereof or a variant, modified form, homologue or derivativethereof.

In particular, the disclosure provides a plasmid or vector systemcomprising a nucleic acid sequence encoding a polypeptide as describedherein or a homologue or derivative thereof.

When compared with human wildtype granzyme B serine protease,polypeptides of the disclosure are characterized inter alia by a lowerinhibition of Serpin B9 and a higher apoptosis inducing potential inmammalian cells. In particular, the serine protease variants accordingto the present disclosure showing in cells or cell lines having SerpinB9 expression (P19⁺⁻cells) a greater apoptotic activity compared to wildtype granzyme B due to the reduced sensitivity to theactivity-inhibiting substance Serpin B9.

In an advantageous embodiment, the polypeptide according to the presentdisclosure comprise a serine protease variant of wildtype human granzymeB as shown in SEQ ID NO: 1, having one or more substitution, insertionor deletion at positions selected from the group of positions thatcorrespond structurally or by amino acid sequence homology to thepositions 28 and/or 201, or a modified form thereof, wherein saidpolypeptide having a greater apoptotic activity compared to wildtypegranzyme B.

In particular, the polypeptide according to the present disclosurecomprises a serine protease variant of wildtype human granzyme B (GB), aserine-dependent and aspartate-specific protease, or a derivativethereof. Granzyme B is a component of cellular immune defense which,upon activation of cytotoxic T cells (CTL) or natural killer cells (NK),is secreted from the cytotoxic granules of these cells (Kam, C. M. etal. 2000; Shresta, S. et al. 1998). Upon the perforin-dependenttranslocation of granzyme B into the cytoplasm of attacked cells, aproteolytic cascade is initiated which ends in the apoptosis of thetarget cell (Greenberg, A. H. 1996). The exact function of the perforinsecreted along with granzyme B is still being discussed currently, butit is not capable of inducing apoptosis alone. In the cell membrane,perforin aggregates into 12-18mers and thereby forms pores of 15-18 nm.Initially, it was considered that granzyme B gets into the cytoplasm ofthe target cells through these pores. However, the 32 kDa proteingranzyme B is too large for such a passage. It is more probable toassume that, after granzyme B has bound to perforin and this complex issuccessively internalized, perforin supports the endosomal release ofgranzyme B (Jans, D. A. et al. 1996). In recent years, various proteinscould be identified which are activated by GB-mediated cleavage aredirectly related to apoptosis. Thus, the GB-caused proteolyticactivation of various procaspases, especially 3 and 8, could bedocumented in vitro (Fernandes-Alnemri, T. et al. 1996; Srinivasula, S.M. et al. 1996); these are counted with the central proteases inapoptosis (Nicholson, D. W. and Thornberry, N. A. 1997). Furthercytotoxic activities are displayed by granzyme B in the nucleus. Afterhaving intruded the cytoplasms of the target cell, granzyme B isrelatively quickly translocated into the nucleus in a caspase-dependentway (Pinkoski, M. J. et al. 2000). There, granzyme B is capable, forexample, of cutting nuclear matrix antigen and poly(ADP-ribose)polymerase (Andrade, F. et al. 1998). A quick apoptosis could beobserved in cells after granzyme B accumulated in the nucleus (Trapani,J. A. et al. 1998; Trapani, J. A. et al. 1998). More recent data provethe initiation of apoptosis through the direct proteolytic cleavage ofBid, a member of the Bcl-2 family having only one BH3 domain. Aftercleavage, the truncated form tBid becomes embedded in the mitochondrialmembrane and depolarizes it. This induces the release of cytochrome cand an apoptosis-inducing factor from the mitochondria into thecytoplasm, which critically accelerated cell death (Sutton, V. R. et al.2000). Further caspase-independent toxic properties of granzyme B couldbe described, the underlying mechanism still being uncleared (Beresford,P. J. et al. 1999; Sarin, A. et al. 1997).

Embodiments of the present disclosure pertains to polypeptidescomprising a serine protease having at least 90 percent identity toamino acids 1-227 of SEQ ID NO: 1, and which, as compared to amino acids1-227 of SEQ I D NO: 1, comprises at least one substitution, insertionor deletion at one or more positions corresponding to position 28 or201, or a modified form thereof.

Further embodiments of the present disclosure pertain to polypeptidescomprising a serine protease having at least 85%, at least 90%, at least95, at least 99 percent identity to amino acids 28-202 of SEQ ID NO: 1,whereby at one or more positions corresponding to position 28 or 201 isa modification. In particular the modification is a substitution and thesubstitution is selected from the group consisting of R201A, R201K andR28K, or any combinations thereof.

In the prior art, granzyme B alleles with different alleles encodingthree amino acid substitutions were identified. Therefore, the sequenceof wildtype granzyme B (SEQ ID NO. 1) comprises all these alleles. Inparticular, the three substitutions could be at positions 35, 74 and227. In some embodiments, the substitutions are selected from the groupconsisting of Q35R, P74A and Y227H. In the coding nucleic acid sequenceof human wildtype granzyme B the substitutions are in exon 2, an A→Gsubstitution resulted in the mutation of glutamine (CAA) 48 (numberingwith reference to Estebanez-Perpina et al., Biol. Chem., Vol. 381, pp.1203-1214, December 2000) to arginine (CGA); in exon 3, a C→Gsubstitution changed proline 88 (CCC) to alanine (GCC); and in exon 5, aT→C substitution altered tyrosine 245 (TAC), the last amino acid in theprotein, to histidine (CAC).

Therefore, in further advantageous embodiments, polypeptides accordingto the present disclosure comprise in additional to the variations atone or more positions corresponding to position 28 or 201 a variation atone or more positions corresponding to position 35, 74 and/or 227corresponding to the position of the amino acid sequence of SEQ IDNO: 1. For example, the variation at position 35 is 35R, the variationat position 74 is 74A and the variation at position 227 is 227H.

In further advantageous embodiments, the polypeptides according to thepresent disclosure comprise a substitution, insertion or deletion is atposition 28 and/or 201. In one embodiment, the polypeptides comprise atleast a substitution at position 28 and/or 201. In advantageousembodiments, the substitution is selected from the group consisting ofR28A, R28E, R28K, R201A, R201E and R201K, in particular at least one ofthe substitutions R201A, R201K or R28K or any combinations thereof.

In a further embodiment the polypeptides according to the presentdisclosure have reduced sensitivity towards activity-inhibitingsubstances.

Examples of activity-inhibiting substance are any substance that worksas inhibitors for proteases, in particular for serine proteases, moreparticular for granzymes. In an advantageous embodiment according to thepresent disclosure, the activity-inhibiting substance is specific forgranzyme B.

In some embodiments, the activity-inhibiting substance inhibits granzymeB activity, inhibits granzyme transcription, inhibits granzymetranslation, increases granzyme degradation, or destabilizes granzyme.In other embodiments, the granzyme inhibitor inhibits granzyme function.The granzyme inhibitor can be a polypeptide, an anti-granzyme antibody,or a small molecule. In some specific embodiments, the polypeptide is aserpin. Serpins are endogenous serine protease inhibitors and someexamples of serpin useful in the context of the present invention areSPI6, P19, PI-6, monocyte neutrophil elastase inhibitor (MNIN), PI-8,and plasminogen activator inhibitor 2 (PAI-2). In an advantageousembodiment, the activity-inhibiting substance is serpin B9 (proteinaseinhibitor 9 (PI-9)). Furthermore, in some embodiments, theactivity-inhibiting substance is a virally encoded serpin, cytokineresponse modifier A (CrmA) of poxviruses and/or a 100 kDa assemblyprotein (Ad5-100K) of a human adenovirus, known as adenovirus type 5.

There is no requirement that the polypeptides of the present disclosurecomprise a full-length native polypeptide sequence of a serine protease,in particular of granzyme B. Rather, the polypeptide can also have asequence that is modified from a native polypeptide sequence using thetechniques known to those of skill in the art and/or taught in thisspecification. In some particular embodiments, the polypeptide is anenzyme variant that comprises a sequence that has serine proteasefunction. Those of ordinary skill in the art will understand that it isbe possible to reduce, increase or decrease the number of amino acids inpolypeptide variants according to the present disclosure, so long as theactive site and activity of the polypeptide having serine proteaseactivity are maintained. For example, there are a wide variety ofvariants that can be prepared to meet the needs according to the presentdisclosure and the teachings of this paragraph and the remainder of thespecification can be used to prepare variants based on a large number ofpolypeptides that have granzyme activity.

Therefore, the disclosure pertains to modified form of the polypeptidesaccording to the present disclosure which has at least a minimumpercentage sequence identity and/or percent homology to the polypeptidesaccording to the present disclosure, wherein the minimum percentidentity and/or homology is at least 75%, at least 80%, at least 85%, atleast 90%, at least 93%, at least 96%, at least 97%, at least 98% or atleast 99%.

The polypeptides having serine protease activity according to thepresent disclosure may, in addition to the serine protease activecenter, comprise a leader segment. Typically, these leader segments willbe positively charged amino acid segments that facilitate proteintranslocation into the cytosol of the cell. Examples of such sequencesinclude, but are not limited to an IG-kappa leader sequence. Of course,it is possible for one of ordinary skill to design and test an almostunlimited number of leader sequences that can be used in the invention.In most cases, these sequences simply require a relatively short segmentof primarily positively charged amino acids. For a general review ofsuch leader sequences, one can review Ford et al. 2001.

Embodiments pertain also to compositions comprising the polypeptides ofthe present disclosure, in particular to pharmaceutical, diagnostic orcosmetic compositions.

The polypeptides according to the present disclosure can be used with a“pharmaceutically acceptable carrier” which includes any and allsolvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art. A pharmaceutically acceptable carrier is preferablyformulated for administration to a human, although in certainembodiments it may be desirable to use a pharmaceutically acceptablecarrier that is formulated for administration to a non-human animal,such as a canine, but which would not be acceptable (e.g., due togovernmental regulations) for administration to a human. Except insofaras any conventional carrier is incompatible with the active ingredient,its use in the therapeutic or pharmaceutical compositions iscontemplated.

The actual dosage amount of a composition of the present inventionadministered to a subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In advantageous embodiments, polypeptide according to the disclosure areused for preparing a medicament for preventing or treating a diseaselike allergy, autoimmune reaction, tissue rejection reaction, or chronicinflammation reaction, preferably cancer.

In another aspect, the disclosure pertains to complexes comprising abinding structure and a polypeptide according to the present disclosure.The complex according to the disclosure can be regarded as aheterologous complex which comprises at least two domains, i.e., oneeffector domain and one binding domain, in particular the complexcomprises a fusion protein including a binding structure and apolypeptide according to the present disclosure.

In some further aspects, the binding structure has a binding activityfor cellular surface structures. For example, the polypeptides may beprovided in complex with a cell targeting moiety that is a moiety thatbinds to and/or is internalized by only a selected population of cellssuch as cells expressing a particular cellular receptor. Such a celltargeting may, for example, comprise an antibody, a growth factor, ahormone, a cytokine, an aptamer or an avimer that binds to a cellsurface protein. As used herein the term antibody may refer to an IgA,IgM, IgE, IgG, a Fab, a F(ab′)2, single chain antibody or paratopepeptide. In certain cases, a cell targeting moiety of the invention maytarget a particular type of cells such as a retinal, endothelial, irisor neuronal cell. In still further aspects a cell targeting moiety ofthe invention may be defined as cancer cell binding moiety.

For example, in certain embodiments, the binding structures as celltargeting moieties for use in the current disclosure are antibodies. Ingeneral the term antibody includes, but is not limited to, polyclonalantibodies, monoclonal antibodies, single chain antibodies, humanizedantibodies, minibodies, dibodies, tribodies as well as antibodyfragments, such as Fab′, Fab, F(ab′)2, single domain antibodies and anymixture thereof, hi some cases it is preferred that the cell targetingmoiety is a single chain antibody (scFv). In a related embodiment, thecell targeting domain may be an avimer polypeptide. Therefore, incertain cases the cell targeting constructs of the invention are fusionproteins comprising a polypeptide according to the present disclosureand a scFv or an avimer. In some very specific embodiments the celltargeting construct is a fusion protein comprising a polypeptideaccording to the present disclosure fused to a single chain antibody.

In certain aspects of the disclosure, a binding structure may be agrowth factor. For example, transforming growth factor, epidermal growthfactor, insulin-like growth factor, fibroblast growth factor, Blymphocyte stimulator (BLyS), heregulin, platelet-derived growth factor,vascular endothelial growth factor (VEGF), or hypoxia inducible factormay be used as a cell targeting moiety according to the disclosure.These growth factors enable the targeting of constructs to cells thatexpress the cognate growth factor receptors.

In further aspects of the invention, a binding structure may be ahormone. Some examples of hormones for use in the disclosure include,but are not limited to, human chorionic gonadotropin, gonadotropinreleasing hormone, an androgen, an estrogen, thyroid-stimulatinghormone, follicle-stimulating hormone, luteinizing hormone, prolactin,growth hormone, adrenocorticotropic hormone, antidiuretic hormone,oxytocin, thyrotropin-releasing hormone, growth hormone releasinghormone, corticotropin-releasing hormone, somatostatin, dopamine,melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoids,mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin,glucagon, amylin, erythropoitin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, leptin,thrombopoietin, angiotensinogen, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36.

As discussed above targeting constructs that comprise a hormone enablemethod of targeting cell populations that comprise extracellularreceptors for the indicated hormone.

In yet further embodiments of the invention, binding structures may becytokines. For example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9,IL10, ILI11, IL12, IL13, IL14, IL15, IL-16, IL-17, IL-18,granulocyte-colony stimulating factor, macrophage-colony stimulatingfactor, granulocyte-macrophage colony stimulating factor, leukemiainhibitory factor, erythropoietin, granulocyte macrophage colonystimulating factor, oncostatin M, leukemia inhibitory factor, IFN-GAMMA,IFN-ALPHA, IFN-βETA, LT-BETA, CD40 ligand, Fas ligand, CD27 ligand, CD30ligand, 4-1BBL, TGF-BETA, IL Ialpha, IL-I BETA, IL-1RA (Interleukin 1receptor antagonist), MIF and IGIF (IFN-gamma inducing factor) may allbe used as targeting moieties according to the disclosure.

In certain aspects of the disclosure the binding structure may be acell-targeting moiety, in particular a cancer cell-targeting moiety. Itis well known that certain types of cancer cells aberrantly expresssurface molecules that are unique as compared to surrounding tissue.Thus, cell-targeting moieties that bind to these surface moleculesenable the targeted delivery of the polypeptides of the presentdisclosure specifically to the cancers cells. For example, a celltargeting moiety may bind to and be internalized by a lung, breast,brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck,esophageal, liver, skin, kidney, leukemia, bone, testicular, colon orbladder cancer cell. The skilled artisan will understand that theeffectiveness of cancer cell targeted polypeptides of the presentdisclosure may, in some cases, be contingent upon the expression orexpression level of a particular cancer marker on the cancer cell. Thus,in certain aspects there is provided a method for treating a cancer withtargeted polypeptides of the present disclosure comprising determiningwhether (or to what extent) the cancer cell expresses a particular cellsurface marker and administering polypeptide targeted therapy (oranother anticancer therapy) to the cancer cells depending on theexpression level of a marker gene or polypeptide.

In advantageous embodiments, the binding structure of the complexbelongs to the group of antigen binding polypeptides/proteins targetingcell type specific markers, in particular the binding structure isdirected against cancer cell specific structures, disease specificstructures of pathogenic substances or pathogenic matter or the bindingstructure is binding to soluble markers of disease/environment/food andfeed safety or biodefense.

In particular, in advantageous embodiments the binding structure of thecomplex comprises moieties which are affinity moieties from affinitysubstances or affinity substances in their entirety selected from thegroup consisting of antibodies, antibody fragments, receptor ligands,enzyme substrates, lectins, cytokines, lymphokines, interleukins,angiogenic or virulence factors, allergens, peptidic allergens,recombinant allergens, allergen-idiotypical antibodies,autoimmune-provoking structures, tissue-rejection-inducing structures,immunoglobulin constant regions and their derivatives, mutants orcombinations thereof. In further advantageous embodiments, the antibodyfragment is a Fab, an scFv; a single domain, or a fragment thereof, abis scFv, Fab2, Fab3, minibody, maxibody, diabody, triabody, tetrabodyor tandab, in particular a single-chain variable fragment (scFv). Forexample, the scFv is specific for the CD64 receptor and/or for the CD30receptor.

In some embodiments, the complex has one or more supplementarycomponents S in addition to the binding structure and a polypeptideaccording to the present disclosure. From his former experience, theskilled person knows that additional features and properties can have acritical importance to the efficient preparation and/or effectiveness ofthe complexes according to the invention. Due to the distinctness of thediseases to be treated with the complexes according to the disclosure,an adaptation of the complexes to the respective particularcircumstances may be necessary.

The component S may be selected from the group consisting of aninducible promoter capable of regulating synthetic performance, a leadersequence capable of controlling protein biosynthesis, His tag, affinitytag, translocation domain amphiphatic sequence capable of translocatingan apoptotic agent into a target cell, and a synthetic pro-granzyme Bamphiphatic sequence capable of intracellular activation of a granzyme.

In advantageous embodiments, the component S is a leader sequence forsecretory expression and/or the component S is a enterokinase cleavagesite enabling activation of a polypeptide according to the presentdisclosure and/or the component S is a HIS tag or affinity tag, enablingpurification of the complex.

In a further embodiment of the invention there is provided an isolatednucleic acid sequence comprising sequence encoding a polypeptide asdescribed supra. Thus, a nucleic acid sequence encoding any of thepolypeptides or polypeptide fusion proteins described herein are alsoincluded as part of the instant invention. The skilled artisan willunderstand that a variety of nucleic acid sequence may be used to encodeidentical polypeptides in view of the degeneracy of genetic code. Incertain cases for example the codon encoding any particular amino acidmay be altered to improve cellular expression.

In preferred aspects, a nucleic acid sequence encoding a polypeptide ofthis disclosure is comprised in an expression cassette. As used hereinthe term “expression cassette” means that additional nucleic acidssequences are included that enable expression of the polypeptides in acell, or more particularly in a eukaryotic cell. Such additionalsequences may, for examples, comprise a promoter, an enhancer, intronsequences or a polyadenylation signal sequence.

In still further aspects of the disclosure a coding sequence for thepolypeptides may be comprised in an expression vector such as a viralexpression vector. Viral expression vectors for use according to theinvention include but are not limited to adenovirus, adeno-associatedvirus, herpes virus, SV-40, retrovirus and vaccinia virus vectorsystems.

Thus, in a specific embodiment, there is provided a method for treatinga patient with cancer comprising administering to the patient aneffective amount of a therapeutic composition comprising a polypeptideaccording to the present disclosure or a nucleic acid expression vectorencoding a polypeptide as described supra. In preferred aspects, methodsdescribed herein may be used to treat a human patient.

As described above, in certain aspects, the disclosure provides methodsfor treating cancer. Thus, in certain cases, described methods may beused to limit or reduce tumor cells by apoptosis thereby reducing tumorgrowth or metastasis. A variety of cancer types may be treated withmethods of the present disclosure, for example a cancer for treatmentmay be a bladder, blood, bone, bone marrow, brain, breast, colon,esophagus, eye, gastrointestinal, gum, head, kidney, liver, lung,nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, oruterus cancer. Furthermore additional anticancer therapies may be usedin combination or in conjunction with methods of the invention. Suchadditional therapies may be administered before, after or concomitantlywith methods of the disclosure. For example an additional anticancertherapy may be chemotherapy, surgical therapy, an immunotherapy or aradiation therapy.

It is contemplated that polypeptides, compositions and/or complexes ofthe disclosure may be administered to a patient locally or systemically.For example, methods of the invention may involve administering acomposition topically, intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intraocularly,intranasally, intravitreally, intravaginally, intrarectally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, by inhalation, by injection, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, via a catheter, or via a lavage.

Further, embodiments of this disclosure relate to the use of thepolypeptides, compositions and complexes according to the presentdisclosure for the preparation of a pharmaceutical, diagnostic orcosmetic composition. In some embodiments, the disclosure pertains tomedicaments comprising the complex of the disclosure in combination witha pharmacologically acceptable carrier or diluent as defined above.

In another aspect, the disclosure relates to methods of treating amalignant disease, an allergy, autoimmune reaction, tissue rejectionreaction, or chronic inflammation reaction comprising administering aneffective amount of the complex according to the present disclosure to apatient in need thereof.

For the example of the anti-CD30 apoptotic agent Ki4(scFv)-granzyme BR28K (Gb-Ki4(scFv) R28K) and Ki4(scFv)-granzyme B R201K (Gb-Ki4(scFv)R201K), the cytotoxic effectiveness of complexes based on the presentdisclosure could be proven for the example of the cell line L1236. Thesecretion of this functional complex from eukaryotic cells additionallydemonstrates the potential suitability of the proteins according to theinvention for a gene-therapeutic application.

Cellular compartments or host cells which synthesize complete complexesaccording to the disclosure or individual components thereof aftertransformation or transfection with the nucleic acid molecules orvectors according to the invention are also claimed according to theinvention.

The cellular compartments or host cells according to the disclosure areof either prokaryotic origin, especially from E. coli, B. subtilis, S.camosus, S. coelicolor, Marinococcus sp., or eukaryotic origin,especially from Saccharomyces sp., Aspergillus sp., Spodoptera sp., P.pastoris, primary or cultivated mammal cells, eukaryotic cell lines(e.g., CHO, Cos or 293) or plant systems (e.g. N. tabacum).

The following methods and examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present disclosurein any way.

METHODS AND EXAMPLES

In the following examples, materials and methods of the presentdisclosure are provided including the determination of catalyticproperties of enzymes obtained by the method. It should be understoodthat these examples are for illustrative purpose only and are not to beconstrued as limiting this disclosure in any manner. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

Example 1 Recombinant Techniques for Manufacturing a Complex ComprisingGranzyme B Variants

The construction of the pMS plasmids encoding the sequence ofGb-H22(scFv) has already been described (Stahnke et al., 2008). Theconstruction of the Gb-Ki4(scFv) encoding plasmid has been cloned beforeas well in a comparable manner. In all FIGS., Ki4 and Ki4(scFv) is theabbreviation for Ki4(scFv).

Site directed mutagenisis was conducted by overlap extension PCR usingspecific primers during an SOE PCR. The primer pairs are shown in table3 below with the corresponding mutations indicated.

TABLE 3 Mutagenesis Primer Variant 5′ Primer ID 5′ Primer Sequence 3′Primer ID 3′ Primer Sequence R28K SEQ ID NO: cagaagtctc tgaag aag tgSEQ ID NO: ggaagccacc gca ctt cttc 10 cggtggcttc c 11 agagacttct g R28ASEQ ID NO: cagaagtctc tgaag gcg tg SEQ ID NO: ggaagccacc gca cgc cttc 12cggtggcttc c 13 agagacttct g R28E SEQ ID NO: cagaagtctc tgaag gag tgSEQ ID NO: ggaagccacc gca ctc cttc 14 cggtggcttc c 14 agagacttct g R201KSEQ ID NO: ctcctatgga  aag aacaatg SEQ ID NO: ggaggcatgc cattgtt ctt 16gcatgcctcc 17 tccataggag R201A SEQ ID NO: ctcctatgga  gca aacaatgSEQ ID NO: ggaggcatgc cattgtt tgc 18 gcatgcctcc 19 tccataggag 30 R201ESEQ ID NO: ctcctatgga  gag aacaatg SEQ ID NO: ggaggcatgc cattgtt ctc 20gcatgcctcc 21 tccataggag

In the same manner the mutated Granzyme B variant K27A was generatedwhich has been described before to have a lower affinity to PI9 (Sun,Whisstock et al. 2001).

The specificity of the binding partners of the cytolytic fusion proteinsare as follows: H22(scFv) is a humanized single chain specific to Fcgamma rexceptor I (CD64) whereas the murine Ki4(scFv) binds to CD30. The72 kDa glycoprotein CD64 (FcγRI) is the mediator of endocytosis andphagocytosis, antibody-dependent cellular cytotoxicity and production ofcytokines and superoxide. It is involved in inflammatory diseases andalso over-expressed on the surface of leukemic cells. CD30 is aglycosylated type I transmembrane protein and belongs to the tumornecrosis factor receptor superfamily. It turned out to be a promisingtarget for the treatment of Hodgkin lymphoma in previous studies(Schwab, Stein et al. 1982; Gruss, Pinto et al. 1996).

FIG. 10A shows the amino acid sequence of granzyme B wildtype accordingto NCBI (AAA75490.1 GI: 181186) with the allele Q35R (or Q48R dependingon numbering of amino acids; also compare RAH mutations due to differentalleles described in Sun et al.: “Granzyme B encoded by the commonlyoccurring human RAH allele retains pro-apoptotic activity”, 2004, TheJournal of biological Chemistry, 279 (17), pp. 16907-16911).

Examples for fusion proteins comprising a granzyme B variant are shownin FIG. 1A and FIG. 1B. Both fusion proteins comprise pCMV as aconstitutive CMV promoter; Igκ as a eader sequence for secretoryexpression, ECS as a Enterokinase cleavage site for the in vitroactivation of granzyme B, a His₆-Tag for affinity purification (H), IRESas an internal ribosome entry site for bicystronic reporter expressionand EGFP as an enhanced green fluorescent protein coding region. Theconstruct in FIG. 1A comprises H22 as a CD64 specific scFv and theconstruct of FIG. 1B comprises Ki4 (for Ki4(scFv)) as a CD30 specificscFv.

Example 2 Cell Lines and Primary Cells

The used cell lines L540, L428, L1236 and K562 and the expression cellline HEK293T (Graham et al., 1977) were kept in RPMI complex medium(RPMI 1640 plus Gluta-MAX-I) supplemented with 10% (v/v) FCS and 100μg/ml penicillin and streptomycin (abbreviated as R10) at 37° C. and 5%CO₂. After transfection of HEK293T cells 100 μg/ml Zeocin was added forselection purposes. L428 (ACC-197), L540 (ACC-72) and L1236 (ACC-530)are Hodgkin derived cell lines. L1236 is established from the peripheralblood of a 34-year-old man with Hodgkin lymphoma (mixed cellularity,stage IV, refractory, terminal, third relapse) in 1994, L428 isestablished from the pleural effusion of a 37-year-old woman withHodgkin lymphoma (stage IVB, nodular sclerosis, refractory, terminal) in1978 and L540 is established from the bone marrow of a 20-year-old womanwith Hodgkin lymphoma (nodular sclerosis; stage IVB, pre-terminalstage). L540cy (von Kalle, Wolf et al. 1992) have been re-cultivatedafter one passage of growth within mice. Their characteristics regardingsensitivity to granzyme B mediated cell death, PI9 expression, CD30receptor expression have been tested to be the same as for L540. K562(ACC-10) is established from the pleural effusion of a 53-year-old womanwith chronic myeloid leukemia (CML) in blast crisis in 1970.

Primary cells from CMML (chronic myelomonocytic leukaemia) patients wereobtained after informed consent and with the approval of the ClinicalResearch Ethics Board of the University of Aachen. Mononuclear cellswere isolated from peripheral blood by density gradient centrifugationusing Biocoll separating solution (Biochrom AG) and cultured in RPMIcomplex medium. For viability assays 200 U/ml Interferon γ was added forstimulation of CD64 expression.

Example 3 Detection of Serpin B9 in Tumour Cell Lines and Primary TumourCells Via Western Blot Analysis and Flow Cytometry

For the detection of endogenous Serpin B9 expression within tumor celllines or primary tumour cells from leukemic patients, 10⁶ cells werelysed within 50 μl lysis buffer (Phosphate buffered Saline (PBS)supplemented with 1% Triton X-100) for 30 minutes on ice. The celllysate was cleared via centrifugation and the protein concentrationdetermined with Bradford reagent (BioRad). 40 μg of total solubleprotein was loaded on an SDS gel for western blot analysis. Afterelectroblotting onto nitrocellulose membranes and blocking with PBSTcontaining 2.5% milk powder, Serpin B9 was detected with anti-P19 (SantaCruz, clone 7D8) and GAM-PO and visualized by an enhancedchemoluminescence (ECL) system (BD BioScience). The membrane was washedwith PBST before development.

In parallel, detection of endogenous Serpin B9 expression was analyzedvia a FACSCalibur flow cytometer (Becton Dickinson). Therefore 10⁶ cellswere washed with PBS and 500 μl Cytofix/Cytoperm (BD BioScience) wasadded in order to fix and permeabilize the cells. After incubation for20 minutes on ice and washing of the cells, a blocking step was includedwith 5% BSA in 200 μl PBS for 20 minutes on ice. The cells were washedonce with PBS+0.2% Tween-20 and incubated with the first antibodyanti-P19 (Santa Cruz, clone 7D8) for 30 minutes on ice. The secondantibody GAM-FITC was added to the washed cells and incubated asmentioned above. The final washing step and resuspension of the cellswas done in PBS+0.2% Tween-20. Thus, with the help of flow cytometricanalysis the endogenous expression of Serpin B9 could be detected. Forevaluation the mean fluorescence intensities (MFI, shift in fluorescenceintensity of a population of cells during flow cytometry) were compared.Mean values and standard deviations could be calculated from threeindependent experiments. For the flow cytometric analysis the programCellQuest Version 3.3 (Becton Dickinson, Heidelberg) was used, forgraphical determinations WinMDI 2.8 (1993-1998 Joseph Trotter) wasapplied.

FIG. 3 shows the results of the determination of endogenous Serpin B9 indifferent cell lines via flow cytometric analysis. The cells wereanalyzed for endogenous Serpin B9 expression as described above. Theprotein expression is the highest in L428 and L1236 compared to controlcell lines Jurkat and L540 (same result for L540cy). The K562 cell lineis also clearly PI9 positive. The measured fluorescence shift wasquantified as MFI (Mean Fluorescence Intensity) and mean values werecalculated (see above). The error bars indicate standard deviation fromthree independent experiments.

FIGS. 4A-B show the results of the determination of Serpin B9 expressionin different cell lines (CD30 positive K562, L540, L428 and L1236 (FIG.4A) and CD64 positive HL60 (FIG. 4B)) via western blot analysis. Thecells were lysed as described above and 40 μg cell lysate was loaded ona 12% SDS gel. Serpin B9 was detected by specific anti-P19 and GAM-POand visualized by ECL. M: Marker in kDa, P: positive control(recombinant PI9 from E. coli). It can be seen that K562, L428 and L1236clearly express Serpin B9 whereby K562 expresses the lowest amount.

FIG. 17C and FIG. 17E show the results of the determination of Serpin B9expression in primary CMML cells via western blot analysis. The cellswere lysed as described above and 40 μg cell lysate was loaded on a 12%SDS gel. Serpin B9 was detected by specific anti-P19 and GAM-PO andvisualized by ECL. M: Marker in kDa. It can be seen that primary cellsalso express the granzyme B inhibitor Serpin B9 during cultivation ofthe cells (days equal cultivation time).

Example 4 Protein Expression in Mammalian Cells and Purification

HEK293T cells were used as expression cell line. The cells weretransfected with 1 μg DNA according to the manufacturer's instructionsusing RotiFect (Roth). The used pMS plasmid encodes for the bicistronicEGFP reporter so that expression of the target protein could be verifiedby the green fluorescence via fluorescence microscopy.

The secreted protein could be purified from the cell culture supernatantvia Immobilized Metal-ion Affinity Chromatography (IMAC) and FastPerformance Liquid Chromatography (FPLC). The cleared supernatant wassupplemented with 10 mM imidazole and loaded to an XK16/20 column(Amersham/GE Healthcare) containing 8 ml Sepharose 6 Fast Flow resin(Clontech/Takara). The used buffers such as incubation, washing andelution buffer were described before (Stocker, Tur et al. 2003). Theeluted protein was re-buffered into 20 mM Tris, pH 7.4, 50 mM NaCl,concentrated, aliquoted and stored at −80° C. For activation prior touse Enterokinase was added to the protein (0.02 U/μg) with 2 mM CaCl₂for 16 h incubation at 23° C. The protein concentration was calculatedfrom Coomassie stained SDS gels using AIDA Image Analyzer Software. Thepurified proteins or cell lysates were analysed via SDS-PAGE underreducing conditions and Coomassie staining. Western blots were performedaccording to standard techniques.

FIGS. 2A-B exemplary show the expression of granzyme B mutants fused tothe binder Ki4(scFv) in HEK293T cells after purification via affinitychromatography. 10 μl of elution fractions were loaded on a SDS-PAGE geland protein was stained with Coomassie (FIG. 2A) or western blot wasperformed with anti-Gb/GAM-PO (FIG. 2B).

Example 5 Expression and Purification of Recombinant Serpin B9 in E.coli

For the expression of recombinant Serpin B9 the E. coli expressionstrain BL21 (DE3) was used for a 4 L fermentation in synthetic medium.The bacteria were harvested 24 h after induction with IsopropylThiogalactoside (IPTG). After centrifugation the bacterial pellet wasre-suspended in lysis buffer (50 mM NaH₂PO₄, pH 8.0, 500 mM NaCl, 0.5 mMDTT) and sonicated six times for 60 s, 70%, 9 cycles. The supernatantwas supplemented with 10 mM imidazol and purified via IMAC as describedabove. After a second affinity purification step, the protein wasre-buffered into 20 mM Tris, pH 7.4, 1 mM DTT and further purified viaanion exchange chromatography (Q-Sepharose XL, 1 ml, GE Healthcare) witha salt gradient of 0.05-1 M NaCl. In order to achieve satisfying purity,size exclusion chromatography (SEC) followed using a Superdex 75 (GEHealthcare) column in 20 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT. Thepurified protein was used for complex formation experiments described inexample 6.

Example 6 Complex Formation and Enzymatic Activity Measurements

The complex formation between recombinant Serpin B9 (see example 5) andGb-H22(scFv) and its mutants took place in a 5:1 molar ratio underreducing conditions in 20 mM Tris, pH 7.4, 50 mM NaCl and 1 mM DTT. 600ng wildtype or mutant Gb-H22(scFv) were incubated with or without SerpinB9 for 1 hour at 37° C. The remaining activity was detected by cleavageof 200 μM of the synthetic substrate Ac-IETD-pNA (Calbiochem/Merck)which mimicks the cleavage site of Pro-caspase 3. The reaction wasmonitored in a microplate reader at 37° C. for one hour with 1 mininterval with an absorbance of 405 nm. The velocity of the activity wascalculated from the linear slope of the reaction in the first 10-12minutes and converted to pmol/min with the help of the correspondingconversion factor (μM/A_(405nm).).

FIG. 6 shows the proteolytic activity of granzyme B variants, fused toH22(scFv) after pre-incubation with Serpin B9. It can be seen that themutants Gb-H22(scFv) R28K and Gb-H22(scFv) R201K remain most of theiractivity in presence of P19. Mean values (for calculation of activitysee above) and standard deviations are based on three independentexperiments.

Example 7 Binding Analysis

The binding of Gb-Ki4(scFv) mutants to the target cell lines L428 andL1236 was evaluated by flow cytometry. 4×10⁵ cells were washed with PBSand incubated with 1 μg purified protein in 100 μl PBS for 30 minutes onice. After 2 wash cycles (Dade Serocent) cells were incubated withanti-His Alexa 488 for 30 min on ice in the dark. Unbound antibodieswere removed by washing with PBS. Specific binding was determined withthe help of FACSCalibur flow cytometer (Becton Dickinson).

FIG. 5 shows the specific binding of Gb-Ki4(scFv) mutants to targetcells. 10⁵ cells were incubated with 1 μg purified protein and detectedwith anti-His Alexa 488. The detection occurred via flow cytometry.Histogram shown exemplary for L428 (representative for L1236 and L540cy)(X: FL1, Y: events).

Example 8 Apoptosis and Viability Assay

Apoptosis was documented via AnnexinV/Propidium iodide (PI) staining.2*10⁵ cell/ml were incubated at 37° C. and 5% CO₂ with 11.1 or 33.3 nMprotein for 24 h, 48 h or 72 h in 12 well plates. After incubation,cells were washed in PBS and the pellet was re-suspended in 450 μlcell-free culture supernatant from HEK293T cells expressingAnnexinV-labelled green fluorescent protein (EGFP, (Stocker, Pardo etal. 2008)) supplemented with 10× AnnexinV binding buffer (100 mM HEPES,pH 7.5, 1.5 M NaCl, 50 mM KCl and 20 mM CaCl₂) as well as 5 μg/ml PI.The incubation took place for 20 minutes on ice in the dark and theanalysis was done via flow cytometric measurements. FL1 channel (X axis)detects GFP fluorescence whereby FL3 channel (Y axis) determines PI.Explanation of corresponding dotplots: quadrant upper right=lateapoptotic cells (AnnexinV and PI positive), quadrant upper left=necroticcells (AnnexinV negative and PI positive), quadrant lower right=earlyapoptotic cells (AnnexinV positive and PI negative), quadrant lowerleft=viable cells (AnnexinV and PI negative).

The cytotoxic effect of Gb-Ki4(scFv) wildtype and its variants on CD30positive cell lines or Gb-H22(scFv) wildtype and its variants onmononuclear cells from CMML patient material was monitored using theability of metabolic active cells to reduce the tetrazolium salt XTT toorange colored compounds of formazan. The intensity of light wasmeasured by a microplate reader and is directly proportional to thenumber of living cells. 2*10⁵ cells were plated in 1 ml of R10 in 12well plates either in 1:5 serial dilutions or with a single proteinconcentration of 11 nM of the respective cytolytic fusion protein andincubated at 37° C. and 5% CO₂. After 48 or 72 hours incubation 100 μlof the cell suspension were transferred into 96 well plates and 50 μlXTT was added. Read out was done at 450 nm with reference wavelength of650 nm.

In order to determine the Caspase 3/7 activity a pre-luminescentCaspase-3/7-DEVD-aminoluciferine substrate was used (CaspaseGlo™ 3/7Assay, Promega). Caspase 3/7 is a direct substrate of Granzyme B. If itis cleaved after Granzyme B delivery into the target cells and therebyactivated, it can cleave the substrate so that free unboundaminoluciferine is released. This is then used by luciferase whereby aluminescence signal is produced. Thus, the measured luminescence isdirectly proportional to the activity of caspase 3/7.

FIGS. 7A-B and FIGS. 8A-C show the results of the apoptosis assay byAnnexinV-GFP staining of Gb-Ki4(scFv) mutants on PI9 positive targetcell line L1236. Results of three independent experiments are shown,either as dotplots (X: FL1, Y:FL3, explained above) or as bar graph (Y:Amount of pre and late apoptotic cells in relation to total cellpopulation seen in dotplot of flow cytometric analysis or viability[%]). (FIG. 7A) 33.3 nM protein was added to cells, incubation for 48 h;(FIG. 7B) 11.1 nM protein was added to cells, incubation for 48 h; (FIG.8A) 33.3 nM protein was added to cells, incubation for 72 h; (FIG. 8B)11.1 nM protein was added to cells, incubation for 72 h. (FIG. 8C) 11.1nM Gb-Ki4(scFv) variants was added to 2*10⁵ cells in 1 ml for 48 hoursat 37° C. Viability was determined via addition of 50 μl XTT to 100 μlcells after transfer into a 96 well plate. Negative control is with PBSbuffer. Standard deviations are shown for 3 to 5 independentexperiments. Statistical significance determined via student t-test:(**) for p<0.05, (***) for p<0.001. Results indicate an increasedcytotoxic effect of the mutants compared to the wildtype and the mutantfrom literature (K27A). The most promising mutants from this example areGb-Ki4(scFv) R28K comprising a modified granzyme B (SEQ ID NO: 1) with asubstitution at position 28 from R to K, Gb-Ki4(scFv) R201A comprising amodified granzyme B (SEQ ID NO: 1) with a substitution at position 201from R to A and Gb-Ki4(scFv) R201K comprising a modified granzyme B (SEQID NO: 1) with a substitution at position 201 from R to K.

FIGS. 9A-C show the results of an apoptosis assay of Gb-Ki4(scFv)mutants on PI9 positive target cell line L428. Verification of apoptosisvia AnnexinV-GFP assay (Clontech). Results of three independentexperiments are shown, either as dotplots (X: FL1, Y:FL3, explainedabove) or as bar graph (Y: sum of pre and late apoptotic cells inrelation to total cell population or viability [%]). (FIG. 9A) 33.3 nMprotein was added to cells, incubation for 24 h; (FIG. 9B) 11.1 nMprotein was added to cells, incubation for 48 h. (FIG. 9C) 11.1 nMGb-Ki4(scFv) variants was added to 2*10⁵ cells in 1 ml for 48 hours at37° C. Viability was determined via addition of 50 μl XTT to 100 μlcells after transfer into a 96 well plate. Negative control is with PBSbuffer. Standard deviations are shown for 3 to 5 independentexperiments. Statistical significance determined via student t-test:(**) for p<0.05 (***) for p<0.001. The most promising mutants are thesame as described above for L1236.

FIG. 15 shows the viability of PI9 positive K562 cells after incubationwith 11.1 nM cytolytic fusion protein for 48 hours at 37° C. Results ofthree independent experiments are shown as bar graph (Y: viability [%]).Gb-Ki4(scFv) R201K leads to the highest apoptotic rates. Viability wasdetermined via addition of 50 μl XTT to 100 μl cells after transfer intoa 96 well plate. Negative control is with PBS buffer. Standarddeviations are shown for 3 to 5 independent experiments. Statisticalsignificance determined via student t-test: (**) for p<0.05 (***) forp<0.001.

FIG. 16 shows the caspase 3/7 activity after 48 hours incubation with11.1 nM cytolytic fusion protein. Results of three independentexperiments are shown as bar graph (Y: relation of luminescence oftreated samples to background signal of Gb-Ki4(scFv) [−]). The R201Ksubstitution resulted in a 22-25% increase in caspase 3/7 activity ofSerpin B9 positive cell lines compared to the wildtype protein.

FIG. 17A-E show results of apoptosis and viability assays on primarycells from leukemic patients and determination of their SerpinB9expression. Results of three independent experiments are shown either asbar graph (Y: apoptotic cells [%] or viability [%]) or as western blot.(FIG. 17A) Results from AnnexinV Assay after incubation with 33 nMcytolytic fusion protein for 24 h. (FIG. 17B) Viability measurement ofcells incubated for 72 hours with 33 nM cytolytic fusion proteindetermined via XTT. (FIG. 17C) Anti-SerpinB9 western blot of the usedcells used in (FIG. 17A) and (FIG. 17B) (see example 2 for descriptionof method). (FIG. 17D) Viability assay with cells from a differentpatient after incubation for 24 h with 33 nM cytolytic fusion protein.(FIG. 17E) Anti-SerpinB9 blot (see example 2) of the used cells from(FIG. 17D). The novel Serpin B9 resistant variants lead to highestapoptotic rates in comparison to the wildtype and the mutant fromliterature (K27A).

Example 9 Mouse Experiments

The experiments were officially approved by the local Animal Care andUse Review Committee. All animals received humane care in accordancewith the requirements of the German Tierschutzgesetz, §8 Abs. 1 and inaccordance with the Guide for the Care and Use of Laboratory Animalspublished by the National Institute of Health.

6- to 8-week-old female BALB/c nu/nu mice (Charles River, Germany) wereused. For injection of PI9 positive tumor cells, L428 transfected withfar red fluorescent protein Kat2 (pTag-Katushka2-N; Evrogen) were usedafter washing with PBS and resuspension in 50% BD Matrigel™ BasementMembrane Matrix High Concentration, Growth Factor Reduced (BDBioscience). 5*10⁶ cells in 30 μl were injected subcutaneously in theright hind limb of 21 mice. Treatment was started one day after cellinjection since previous experiments demonstrated immediate growth ofthe cells within the first week after injection. The mice wererandomized into three groups of seven animals and obtained daily dosesof 50 μg Gb-H22(scFv) R201K, Gb-Ki4(scFv) or Gb-Ki4(scFv) R201Krespectively for 5 days. At the same time tumor growth measurements wereperformed via imaging as shown in former studies (Pardo, Stocker et al.2012) with the CRi Maestro system (Cri Inc., Woburn, Mass., USA). Imageswere taken and analysed with the Maestro Spectral Imaging Software. Inorder to monitor the Kat2 signal of the transfected L428 cells, theyellow filter set (630-850 nm) was used. Treatment response withGb-Ki4(scFv) and Gb-Ki4(scFv) R201K was compared to the non-specificcontrol group treated with Gb-H22(scFv) using one-tailed t-test (GraphPad Prism, Version 4.0c).

For injection of PI9 negative tumor cells, L540cy were used afterwashing with PBS and resuspension in 50% BD Matrigel™ Basement MembraneMatrix High Concentration, Growth Factor Reduced (BD Bioscience). 5*10⁶cells were injected subcutaneously in a volume of 30 μl into the righthind limb of the mice. Treatment was started when tumor size was 3 to 5mm. Tumor size was determined via triplicate caliper measurements. Themice were randomized into two groups and obtained 50 μg Gb-H22(scFv)R201K (n=3) or Gb-Ki4(scFv) R201K (n=6) every day for 5 days, afterwardsin 2-days intervals. Treatment response with Gb-Ki4(scFv) R201K wascompared to the unspecific Gb-H22(scFv) R201K control group usingone-tailed t-test (GraphPad Prism, Version 4.0c). p<0.05 was consideredto be statistically significant.

FIGS. 18A-B show the results of mouse experiments: Tumor reduction aftertreatment with cytolytic fusion protein: black triangles: Gb-Ki4(scFv)R201K, grey triangles Gb-Ki4(scFv), grey squares Gb-H22(scFv) R201K. Yaxis shows tumor size in %, X axis shows duration of experiment in days.A) Result for Kat2 transfected L428 tumors (fluorescent): 50 μg proteinwas injected intravenously daily for 5 days, starting one day afterinjection of cells. The tumor growth was calculated with the Cri MaestroSystem every day during treatment cycle. The tumor size on day 1 was setto 100%. The decelerated tumor growth of the group treated with themutant compared to the unspecific control was confirmed statisticallysignificant (*** p<0.001). The difference between treatment with mutantand wildtype was statistically different with p<0.05 (**) except for day2. B) Result for L540cy tumors: 50 μg protein was injected intravenouslydaily 5 for days, then in 2-days intervals until day 13 of theexperiment. The tumor growth was measured via caliper in triplets. Thedifference between the curves was determined to be statisticallysignificant (p<0.05). These results indicate that cytolytic fusionproteins based on the Granzyme B mutant R201K kills PI9 positive tumourcells in vivo whereby wildtype version does not lead to a decrease intumor growth. Also on the PI9 negative cells L540cy, the mutant isfunctional and kills those cells in vivo as well.

In summary, the present disclosure pertains in advantageous embodimentsto polypeptides comprising a serine protease variant of wild type humangranzyme B as shown in SEQ ID NO: 1, having a modification at one ormore positions selected from the group of positions that correspondstructurally or by amino acid sequence homology to the positions 28and/or 201, or variants, modified forms, homologs, fusion proteins,functional equivalents or functional fragments thereof, wherein saidpolypeptide having a greater apoptotic activity compared to wild typegranzyme B.

The polypeptides of the present disclosure have one or more of thefollowing characteristics:

-   -   The functional fragment has at least 85%, at least 90%, at least        95 or at least 99% identity to amino acids 28-202 of SEQ ID NO:        1.    -   The modification is a substitution, insertion, deletion,        phosphorylation, acetylation like palmitoylation, methylation,        sulphation, glycosylation, lipidation like isoprenylation,        farnesylation, attachment of a fatty acid moiety, glypiation and        ubiquitinylation, and/or    -   The modification is at position 28 and/or 201, and/or    -   The modification is a substitution.    -   The modification is a conservative substitution.    -   The modification is is a neutral substitution.    -   The modification is at least a substitution at position 28        and/or 201.    -   The substitution is selected from the group consisting of R28A,        R28E, R28K, R201A, R201E and R201K.    -   The polypeptides comprise at least one of the substitutions        R201A or R201K and/or R28K, or any combinations thereof.    -   The polypeptide further comprises a substitution of one or more        residues corresponding to position 35, 74 and/or 227.    -   The substitution is selected from the group 35R, 74A and 227H,        or any combinations thereof.    -   The modified form of the polypeptide has at least a minimum        percentage sequence identity and/or percent homology to        polypeptide of claims 1 to 11, wherein the minimum percent        identity and/or homology is at least 75%, at least 80%, at least        85%, at least 90%, at least 93%, at least 96%, at least 97%, at        least 98% or at least 99%.

Furthermore, the present disclosure pertains in advantageous embodimentsto nucleic acid molecules, selected from the group consisting of

-   -   a) a nucleic acid molecule encoding a polypeptide according to        the present disclosure;    -   b) a nucleic acid molecule encoding for a modified form of the        polypeptide according to the present disclosure, preferably in        which one or more amino acid residues are conservatively        substituted;    -   c) a nucleic acid molecule that is a fraction, variant,        homologue, derivative, or fragment of the nucleic acid molecule        presented as SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5;    -   d) a nucleic acid molecule that is capable of hybridizing to any        of the nucleic acid molecules of a)-c) under stringent        conditions    -   e) a nucleic acid molecule that is capable of hybridizing to the        complement of any of the nucleic acid molecules of a)-d) under        stringent conditions    -   f) a nucleic acid molecule having a sequence identity of at        least 95% with any of the nucleic acid molecules of a)-e) and        encoding for a serine protease,    -   g) a nucleic acid molecule having a sequence identity of at        least 70% with any of the nucleic acid molecules of a)-f) and        encoding for a serine protease,    -   h) or a complement of any of the nucleic acid molecules of        a)-g).

Furthermore, the present disclosure pertains in advantageous embodimentsto vectors comprising a nucleic acid molecule according to the presentdisclosure and to host cells being transformed with a vector accordingto the present disclosure and/or comprising a nucleic acid moleculeaccording to the present disclosure.

The present disclosure pertains in advantageous embodiments to isolatednucleic acid molecules selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO: 5, and methods for preparing apolypeptide according to the present disclosure, which comprisesculturing a host cell according to the present disclosure and isolatingthe polypeptide from the culture.

Further, the present disclosure pertains in advantageous embodiments tocompositions comprising a polypeptide according to the presentdisclosure, wherein the compositions may be pharmaceutical, diagnosticor cosmetic compositions and uses of polypeptides according to thepresent disclosure for preventing or treating a disease like allergy,autoimmune reaction, tissue rejection reaction, or chronic inflammationreaction, and in particular cancer.

Furthermore, the present disclosure pertains in advantageous embodimentsto purified complexes comprising a binding structure and a polypeptideaccording to the present disclosure, wherein a complex has one or moreof the following characteristics:

-   -   The complex comprises a fusion protein including a binding        structure and a polypeptide according to the present disclosure.    -   The binding structure belongs to the group of antigen binding        polypeptides/proteins targeting cell type specific markers, in        particular the binding structure is directed against cancer cell        specific structures, disease specific structures of pathogenic        substances or pathogenic matter.    -   The binding structure comprises moieties which are affinity        moieties from affinity substances or affinity substances in        their entirety selected from the group consisting of antibodies,        antibody fragments, receptor ligands, enzyme substrates,        lectins, cytokines, lymphokines, interleukins, angiogenic or        virulence factors, allergens, peptidic allergens, recombinant        allergens, allergen-idiotypical antibodies, autoimmune-provoking        structures, tissue-rejection-inducing structures, immunoglobulin        constant regions and their derivatives, mutants or combinations        thereof.    -   The binding structure is an antibody or an antibody fragment        selected from the group consisting of Fab, scFv; single domain,        or a fragment thereof, bis scFv, Fab₂, Fab₃, minibody, diabody,        maxibody, triabody, tetrabody and tandab, in particular the        binding structure is a single-chain variable fragment (scFv), in        particular specific for CD64 receptor and/or for CD30 receptor.    -   The purified complex according to according to the present        disclosure further comprising at least one component S selected        from the group consisting of an inducible promoter capable of        regulating synthetic performance, a leader sequence capable of        controlling protein biosynthesis, His tag, affinity tag,        translocation domain amphiphatic sequence capable of        translocating an apoptotic agent into a target cell, and a        synthetic pro-granzyme B amphiphatic sequence capable of        intracellular activation of a granzyme.    -   The component S is a leader sequence for secretory expression.    -   The component S is an enterokinase cleavage site enabling        activation of the polypeptide according to the present        disclosure.    -   The component S is an inducible promoter capable of regulating        synthetic performance.    -   The component S is a HIS tag or affinity tag, enabling        purification of the complex.    -   The complex comprises a single-chain variable fragment, a leader        sequence for secretory expression, an enterokinase cleavage        site, an inducible promoter, a HIS tag and a polypeptide        according to the present disclosure.

Further, the the present disclosure pertains in advantageous embodimentsto:

-   -   Nucleic acid molecules coding for the complex according to        according to the present disclosure.    -   Vectors carrying a nucleic acid molecule encoding a complex        according to the present disclosure.    -   Cells transfected with the vector carrying a nucleic acid        molecule encoding a complex according to the present disclosure.

Furthermore, the the present disclosure pertains in advantageousembodiments to:

-   -   Medicaments comprising the complex according to the present        disclosure in combination with a pharmacologically acceptable        carrier or diluent.    -   Methods of treating a malignant disease, an allergy, autoimmune        reaction, tissue rejection reaction, or chronic inflammation        reaction comprising administering an effective amount of the        complex according to the present disclosure to a patient in need        thereof.

What is claimed is:
 1. A polypeptide comprising a serine proteasevariant of the human granzyme B set forth by SEQ ID NO: 1, wherein theserine protease variant has at least 95% identity to SEQ ID NO: 1 andhas a substitution at the position that corresponds structurally or byamino acid sequence homology to position Arg201 of SEQ ID NO: 1, andwherein the serine protease variant has activity to cleave the motifIle-Glu-Thr-Asp.
 2. The polypeptide according to claim 1, wherein theArg201 amino acid is substituted for a different basic amino acid. 3.The polypeptide according to claim 1, wherein the serine proteasevariant is as set forth by SEQ ID NO:
 8. 4. The polypeptide according toclaim 1, wherein the serine protease variant is as set forth by SEQ IDNO:
 9. 5. The polypeptide according to claim 1, wherein the polypeptidefurther comprises a substitution of one or more residues correspondingto position 35, 74 and/or 227 of SEQ ID NO:
 1. 6. An isolated nucleicacid molecule encoding a polypeptide according to claim
 1. 7. Acomposition comprising the polypeptide according to claim 1 in apharmaceutically acceptable carrier.
 8. A purified complex comprising abinding structure and a polypeptide according to claim
 1. 9. Thepurified complex according to claim 8, wherein the complex furthercomprises a fusion protein, wherein the binding structure is an antigenbinding polypeptide or protein that binds a cell type specific marker.10. The purified complex according to claim 8, wherein the bindingstructure is an antibody or an antibody fragment selected from the groupconsisting of Fab, scFv; single domain, or a fragment thereof, bis scFv,Fab₂, Fab₃, minibody, diabody, triabody, tetrabody and tandab.